Readable probe array for in vivo use

ABSTRACT

A disposable high density optically readable polydeoxynucleotide array with integral fluorescence excitation and fluorescence emission channels is described. The compact array size allows integration into several types of interventional devices such as catheters, guidewires, needles, trocars and may be used intraoperatively. Highly sensitive monitoring of the metabolic and disease pathways of cells in vivo under varying chemical, genetic and environmental conditions is afforded.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to applicationSer. No. 60/071,906, filed Jan. 20, 1998.

BACKGROUND OF THE INVENTION

Polydeoxynucleotide and oligonucleotide sequencing with laboratory-basedinstruments has become inexpensive and reliable due to the variety andavailability of complimentary fluorescent labeled target sequences.These fluorescent labeled probes may be specially tailored to hybridizewith genomic DNA segments and form base pair matches that can accuratelydetect the presence of inherited genetic disorders or native-cellmutations. Under excitation light in the visible or UV range, theassociated fluorescent marker attached to the probe emits a secondaryemission which may be detected by a charge-coupled device (CCD) array,photodiode, or other spectrally sensitive light detector.

However, current techniques require the use of specialized reagents andadditional processing to separate the cell wall and other componentsbefore analysis. The analyte is removed and introduced into an assaychamber for analysis. The chambers are housed in portable or tabletopanalytic instruments that typically contain an excitation source,detection sensors, spatial reading or imaging devices, and archivingcapabilities. These systems are expensive and require that tissuesamples be processed prior to use. The biggest drawback to these typesof systems is their inherent inability to perform fast, localizedreading of array probes in a convenient, and repeatable manner in vivo.In vivo monitoring and detection of changes to the human body inresponse to therapy is needed to expedite trials and to monitor resultsfrom therapy, and would allow doctors to treat serious diseases such ascancer safely in a more effective and less costly manner.

SUMMARY OF THE INVENTION

The present invention performs specific detection and analysis ofbiological analytes in vivo using a simplified, low cost set ofcomponents. In one embodiment the small size and simplified operationallows the entire device to be housed in a catheter. In one aspect, thedevice consists of a housing, a light excitation source, and detectorand at least one fluorescent labeled probe material on a substrate thatis exposed to the tissue of the body. The excitation source may bedirected at the substrate carrying the probe, or may be a conductor ofthe excitation energy. Other embodiments include the use of a lumen tointroduce a lysing agent or energy to the area of interest. The lysingagent or energy may be an ultrasonic transducer capable of rupturingcell membranes through the use of a brief burst of ultrasonic energy. Inanother aspect, a lysing system is used in which pressurization andevacuation of the sample via the lumen adjacent to the probe arraycreates a pressure capable of rupturing the cell membrane. Each of theprobes may be read by application of electrical current to theexcitation source and by detecting the presence or absence of signal viathe probe sensor. The probe sensor may be a photodiode that isresponsive to light emitted by the fluorescent probe material. Twoprobes may be mixed and read by two sensors if the spectrum issufficiently separated. A ratio can then be obtained to facilitateanalysis. In another embodiment, a normalizing patch may be adjacent toprovide a reference signal, thereby simplifying the calibration of theinstrument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a planar view of a probe array containing a multiplicity offluorescent probes on its surface.

FIG. 1A is a cross sectional view of the probe array of FIG. 1.

FIG. 1B is a cross sectional view of a sheet of material carrying aprobe array.

FIG. 2 is a cross-sectional view of a readable polydeoxynucleotide arraymodule. (RPAM)

FIG. 2A is a block diagram of the readable polydeoxynucleotide arraymodule and system.

FIG. 3 is a cross sectional view of an interventional device carryingthe readable polydeoxynucleotide array module.

FIG. 4 is a cross sectional view of an interventional device fitted witha lysing core.

FIG. 5 is a side view of a secondary insertable device having a tip anda multifilar shaft.

FIG. 6 is a cross sectional view of a hollow needle carrying thereadable polydeoxynucleotide array module equipped insertable appliance.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring now to FIG. 1, the planar view of a probe array 11 is shown asa grid-like array with a plurality of chambers 13 arranged to haveseparators 15 within a frame 17. The frame 17 may be a smallinjection-molded component made of a plastic such as polystyrene or amolded material such as glass. The separators 15 may be moldedintegrally to the frame 17 or may be separate elements placed within it.The overall dimensions of the frame 17 may be small. Typical dimensionsare less than 1 mm by 1 mm.

Referring now to FIG. 1A, which is a cross sectional view of the probearray 11, the aforementioned separators 15 are effective to separate afluorescent probe material 21 that may have different characteristicsfrom an adjacent fluorescent probe material 23. Probe materials 21 and23 are generally deposited in a thin layer on top of a substrate, inthis case the material of the frame 17. Alternatively, the frame 17 maybe made of a foraminous material or a partly foraminous substance suchas sol gel (not shown). The probe materials may be incorporated into thesubstrate, which may be a flat surface which allows ink printingprocesses to be used to deposit the probe array materials at high speedsand at low cost.

Probe materials generally are engineered molecular materials that aredesigned to have an affinity to one or more constituents that may beexpected to be found in the tissue, fluid or chemical mix to beanalyzed. These probe materials may be made sensitive to specific genesor gene segments through complimentary genetic indicators that have beendesigned to fluoresce or change color, as observed by the naked eye orby spectrographic analysis methods, when they are linked to a moleculeto which they have affinity. A large number of different types andcombinations of optically readable probes are being manufactured todaythat have specific affinity to one or more genes, proteins or otherchemicals. In preferred embodiments, the present invention contemplatesthe use of two classes of probes: (i) protein sensitive probes, such asGFP (green fluorescent probe) from the jellyfish Aequorea Victoria; and(ii) modified oligonucleotide probes that are fluorogenic, such as thosemanufactured by Synthegen LLC, Houston, Tex. 77042. Additional probessuited for use in the present invention are available from MidlandCertified Reagent Company, Midland, Tex. 79701, and Transbio Corp.,Baltimore, Md. 21220. Typically these probes must be used in vitro dueto either their lack of biocompatability or because they must be used inconjunction with aggressive reagents that are toxic to cells.

Various methods and configurations may be used to deposit or arrangeprobe locations and positions in an array or singly. For instance, asheet of plastic material 33, as shown in FIG. 1B, may have lines 35made of probe filled ink printed in any arrangement that may be producedwith printing methods. More than one type of probe-filled ink may beused to produce various patterns and arrangements, including overlappingpatterns (not shown). The ink pattern lines 35 may be protected with atopcoat 37 which may be made of a dissolvable gel such as ordinarygelatin, or another material such as a soluble or even a waterproofpolymer that only dissolves and provides access to the probe material inthe probe-filled ink in lines 35 after the application of a solvent. Thearrangement of the sensitive areas by this process allows the probematerials to be applied to a variety of surfaces and substrates,including medical devices such as needles, trocars, forceps, catheters,guidewires, implants and prostheses, in an inexpensive and reliablemanner.

The following discussion and description of the present invention isdirected to a readable polydeoxynucleotide array module (RPAM). However,those skilled in the art will appreciate that the present invention andspecific embodiments described below may be utilized with any number ofprobe arrays and the RPAM described here is provided as only one,non-limiting, example.

Referring now to FIG. 2, which is a cross sectional view of a readablepolydeoxynucleotide array module (RPAM) 41, the probe array 11 may bepositioned adjacent to a spectrometer module that is encapsulated in anat least partly transparent housing 45. The probe array 11 may becemented to the side, top or other area within a spectrometer module 43with an optical cement (not shown), or by a solvent bond line 47 whichallows two plastics to be fused through partial melting. A spectrometermodule suitable for use in this invention has been described in pendingU.S. patent application Ser. No. 08/898,604, the entire disclosure ofwhich is incorporated by reference herein.

Specifically, the spectrometer module used in the present inventionincludes a light source and a light detector for placement inside a bodysuch that optical conduits are not necessary to deliver light signals toand from the RPAM inside the body. The miniature spectrometer includesthe light source and one or more light detectors. The light sourceilluminates a tissue region and the light detectors detect opticalproperties of the illuminated tissue by measuring modified lightsignals. The light detectors convert optical signals to electricalsignals such that one or more electrical wires placed inside aninterventional device can deliver the electrical signals from the RPAMto a signal display or a microprocessor.

The light source and the light detectors are energized by an externalpower supply through electrical wires. In another embodiment, anoptically transparent tip encapsulates a spectrometer. The tip is shapedto optimize tissue contact and optical transmission. The tipencapsulating the spectrometer is disposed at a distal end of aninterventional device. The tip may be coated with a material to improvelight transmission. The tip may include at least one fluid channel,which is in communication with a lumen inside the interventional device,to deliver a fluid to a tissue region. The spectrometer may also includea light source and the light detectors formed on a single substrate. Thelight source may be a light emitting diode and the light detectors maybe a photodiode comprising multiple channels, where both devices areformed on a silicon substrate. The light detector can include multiplechannels to detect light emission at multiple wavelengths.

Still referring to FIG. 2, probe array 11 may be integrally molded ontothe surface of the spectrometer module 43 creating a somewhat simplifiedone-piece unit which may provide processing advantages in high speedproduction environments where parts counts are intentionally kept low tominimize stock and therefore reduce cost of fabrication and assembly.Injection molding or casting of the components is effective to produceminiature components that correspond in size to conventionalsilicon-based integrated circuit scale. Therefore it should beappreciated that the RPAM may be small, e.g., about the size of aminiature electronic component such as a surface mount device. Suchdevices include packaging, leads, and other components, and may beobtainable in size ranges of less than 1 mm in length. Such devices maytypically be configured in the range from about 0.5 mm to about 3 mm toproduce small, useful devices for in vivo use. The RPAM 41 may also haveprintable surfaces according to the construction of alternative probearray configurations as described in FIG. 1A and FIG. 1B, if desired.Referring once again to FIG. 2, the internal components of the RPAMconsist of a substrate material 49 such as silicon upon which alight-emitting diode light source 51 is mounted with power lead 53attached to one of terminals 55. Various colors and types of diode lightsources may be used, including those now available that emit light inthe infrared, the red, the yellow, the green, the blue, and theblue-violet regions. A working range of RPAM excitation wavelengths isfrom about 1100 nanometers to about 250 nanometers and may comprisemonochromatic, bichromatic or broadband emissions. The exit aperture 57is positioned to illuminate movable mirror 59 which is bonded topiezoelectric stack actuator 61. Empowerment of the stack actuator 61 iseffective to direct light emission from diode light source 51 to one ormore chambers 13. Light emission from the probe materials 21 is pickedup by one or more light detectors 63 through filters 65. Signals fromthe detectors 63 are brought out from the RPAM through other terminals55.

Referring now to FIG. 2A, the operation of the RPAM is depicted in blockdiagram form as follows: Light is generated and directed from lightsource 51 and directed at one or more of chambers 13 by mirror 59, whichimpinges upon at least one probe material 21. Fluorescence or othersecondary light generated by the action of the light energy upon theprobe material causes a second emission that may be detected by one ormore light detectors 63 after passing through a bandpass filter 65. Thesignal may be amplified and/or conditioned by one or more amplifierstages 64. Filters 65 allow the system to discriminate between varioussecondary light emission wavelengths, and signals from said lightdetectors 63 may be synchronized with the operation of light source 51so that at any given time there is a known relationship between theparticular probe that is illuminated and its response as detected by thelight detectors. The timing and relationship of the light generating andlight detecting event and the spatial position of the mirror 59, arecontrolled by CPU 71 and sent to the components via control lines 73.

The data obtained may be stored or presented in a display device orother therapeutic device which can be a graphical display, a televisionmonitor, printout or drug delivery pump, interventional device, motor oractuator, etc. Accordingly, this apparatus may effectively scan or reada plurality of probe materials in a repeatable, fast and controllablemanner, and the information read may be stored, displayed, or used toinitiate another action such as a therapeutic application of a drug, orcontrol of a motor. The bandpass filter system of detecting one or morelight wavelengths for this purpose is basic and that more complexschemes could be employed by those of ordinary skill in the art. Suchschemes may include, without limitation; light wavelength detectionsystems comprising gratings, graduated filters, heterodyne detection,acousto-optic tunable filtering, and other light detectors thateffectively provide and amplitude and frequency responsive signal. Adiffraction grating (not shown), for instance, may be attached tomovable mirror 59 to provide spatial and chromatic controlsimultaneously.

Referring now to FIG. 3, the cross sectional view of an interventionaldevice incorporating the spectrometer and probe still referred to hereas RPAM 41; there is a primary insertable appliance 81 such as acatheter which may have a distal end and a proximal end and may consistof a plastic, rubber or metal material that is generally elongated inshape, has a small cross-section allowing it to pass easily through thebody, and has one or more lumens or conduits which may extend throughthe length of the device. Shown in FIG. 3 is a device having threelumens although a greater or lesser number of lumens may be useddepending upon the application for which the device is intended. Themain lumen 83 is relatively large and is used to deliver a drug, areagent, or a device to or beyond the distal tip 89. Suction lumen 85 isuseful for drawing biological fluids, tissue or other materials intoproximity with the RPAM 41, where the material can be analyzed. Signalwires 74 may extend to an external controller (not shown) or to a CPU,pump, motor or other controller as shown in FIG. 2A, 75.

Returning once again to FIG. 3, infusion lumen 87 may provide additionalfluids, regents, drugs, wires or appliances that may be useful to theprocedure. For example, the practitioner will appreciate that additionalreagents can be introduced to facilitate analysis. Such additionalreagents can include: denaturants, such as guanidinium thiosulfate;buffers, such as Tris-Cl; detergents, such as SDS; chelators, such asEDTA; enzymes, such as proteinases and/or DNAases; and other reagentsknown to those of ordinary skill in the art which may be appropriate tothe particular analysis to be carried out using the apparatus of thepresent invention.

Referring now to FIG. 4, a cross sectional view of an interventionaldevice such as a primary insertable appliance 81 fitted with a lysingcore 101, is shown. The lysing core 101 utilizes mechanical motion todisrupt cells in order to make the cell contents available for analysisby the RPAM (not shown). The use of a lysing device in conjunction withthe RPAM system eliminates the need for potentially toxic reagents thatare commonly used to open cells in vitro. The lysing head 105 consistshere of a more or less hemispherical component that may be comprised ofa metal or plastic, which is mounted at the distal end of a driveshaft103. Such driveshafts are well known for their ability to deliver torqueand rotary motion from a proximal motor 107 or by hand control. Astaught in this invention, motor 107 is one of a class of componentsshown in FIG. 2A as 75 which may be controlled by system CPU 71, alsoshown in FIG. 2A. Numerous other lysing devices are known that mayabrade, disrupt, dissolve, pressurize, vacuum, cavitate or otherwiseapply mechanical forces to a cell or cells that is effective to disruptthe cell and make its contents available for analysis. It should bepointed out that such damage to cells is usually minimized to avoidpermanent damage to the organ, vessel, duct or tissue being tested. Thelysing head 105 need not be relatively large and may be made smallenough so that it may easily pass through the device from the proximalend so that another device or implant may be inserted, if needed,through the same large lumen 83. Such an implant may be a solid orporous, foraminous or dissolvable seed, implant, stent, gel or the like,which may carry therapeutic agents to a particular site in the body.This system provides the advantage that local conditions can bedetermined through use of the polydeoxynucleotide readable array(afforded by the construction of the RPAM device as described herein),and therefore, better and more precise application of appropriatemedicaments, drugs, therapeutic genetically based substances, etc., isfacilitated. Further advantages are provided in that the information isobtained at or near real time, and that information is obtainable fromthe exact location of a proposed therapeutic intervention. Such a devicethat may be used to place an implant is shown in FIG. 5, which is a sideview of a secondary insertable device 111 comprising a rotary,multifilar flexible driveshaft 112 having a therapeutic tip 113terminating in an anchoring device 115 shown as a screw form capable ofbeing screwed into tissue until separable joint 117 breaks, after whichthe remaining part of insertable device 111 may be withdrawn. Driveshaft112 may be hollow, to allow tether 119 to remain attached to therapeutictip 113. Tether material may be constructed of a wire to allow thesending and receiving of an electrical signal, or may simply be used asa retrieval device to retrieve any portion of the therapeutic tip thatmay remain after the need for it is over.

Numerous carrying devices may be used to deliver the RPAM. FIG. 6 is across sectional view of a hollow needle 121 carrying the RPAM insertableappliance 81. The advantage of a needle is that it allows theintroduction of the RPAM into portions of the body where there is nonatural passageway. This method allows the user to position the distaltip of the lysing head 105 in various positions with respect to thesharp needle tip 106. The needle may be of stainless steel and may beinserted into body tissue such as muscle, breast, prostate, or cardiactissue. The needle may be left in place, and the RPAM withdrawntemporarily to allow another appliance (not shown) to be introduced.Other carrying devices may include guidewires, balloon catheters,ultrasound catheters with both imaging or non-imaging, and rotatable orarray configurations, introducer sheaths, balloon angioplasty cathetersfor use in the blood vessels of the heart, the extremities, and thevascular system, atherectomy catheters, and many other types ofinterventional devices, as well as intraoperative devices. The device ofthe invention may be used anywhere there is the need for fast, preciselocalized detection and analysis of nucleotides, proteins or the like,either for diagnostic purposes, or to guide therapy which itself may bemade more localized, and therefore site-specific. Such uses areeconomical and have less impact on surrounding tissue that is free ofdisease. The invention allows use of any agent that may change color asa result of the application of a local chemical to be read and includeswithout limitation such agents as litmus, photodynamic therapeuticagents such as photofrin, fluorescent agents or dyes, staining dyes,luciferin, etc. The present invention permits analysis in a real timefashion without the need to remove and transport tissue specimens forlater analysis.

What is claimed is:
 1. An apparatus comprising: an excitation source; atleast one optically detectable probe directed to an analyte, said probeattached to a substrate and situated to contact said analyte, whereinsaid probe is mixed with an ink to form a probe-filled ink and whereinsaid probe-filled ink is deposited upon said substrate; a detector fordetecting optical properties of said probe, said detector for convertingoptical signals representative of the detected optical properties toelectrical signals; wherein said excitation source, said probe, and saiddetector are adapted for placement together in an area of interestwithin a body.
 2. The apparatus of claim 1 wherein said substratecomprises a sheet of plastic material.
 3. The apparatus of claim 1wherein a plurality of probe-filled inks are deposited upon saidsubstrate in a specific ink pattern.
 4. The apparatus of claim 3 whereinsaid ink pattern is protected by a topcoat.
 5. The apparatus of claim 4wherein said topcoat comprises a dissolvable gel.
 6. The apparatus ofclaim 4 wherein said topcoat comprises a polymer material dissolvableonly upon application of a solvent.
 7. The apparatus of claim 1, whereinsaid excitation source, said probe, and said detector are disposedwithin a body-insertable device that is to be positioned in an area ofinterest within a body.
 8. The apparatus of claim 7, wherein saidbody-insertable device is delivered to the area of interest by acarrying device.
 9. The apparatus of claim 8, wherein said carryingdevice is selected from the group consisting of a hollow needle, a guidewire, a balloon catheter, an ultrasound catheter, an introducer sheath,and a balloon angioplasty catheter.
 10. The apparatus of claim 7 whereinsaid body-insertable device comprises a catheter.
 11. An apparatuscomprising: an excitation source; at least one optically detectableprobe directed to an analyte, said probe situated to contact saidanalyte; a detector for detecting optical properties of said probe, saiddetector for converting optical signals representative of the detectedoptical properties to electrical signals; wherein said excitationsource, said probe, and said detector are disposed within abody-insertable device that is to be positioned in an area of interestwithin a body, said body-insertable device comprising a lumen positionedsuch that said lumen is capable of introducing to said area of interesta lysing system.
 12. The apparatus of claim 11 wherein said lysingsystem comprises an ultrasonic transducer capable of rupturing cellmembranes.
 13. The apparatus of claim 11 wherein said lysing systemcomprises a pressurization and evacuation system capable of rupturing acell membrane.
 14. The apparatus of claim 11 wherein said lysing systemcomprises a mechanical lysing device.
 15. The apparatus of claim 14wherein said mechanical lysing device comprises a lysing head mounted atthe distal end of a driveshaft.
 16. The apparatus of claim 15 whereinsaid driveshaft delivers torque and rotary motion to said lysing headfrom a proximal motor.
 17. The apparatus of claim 11 wherein saidbody-insertable device comprises an implantable device.
 18. Theapparatus of claim 17 wherein said implantable device comprises a rotaryflexible driveshaft having a therapeutic tip terminating in an anchoringdevice.
 19. The apparatus of claim 18 wherein said implantable devicefurther comprises a separable joint between said therapeutic tip suchthat said therapeutic tip remains within a body after removal of saidbody-insertable device.
 20. The apparatus of claim 19 further comprisinga tether such that said tether remains attached to said therapeutic tipafter removal of said body-insertable device.
 21. The apparatus of claim20 wherein said tether is capable of transmitting an electrical signal.22. The apparatus of claim 11 wherein said body-insertable device isdelivered to the area of interest by a carrying device.
 23. Theapparatus of claim 22 wherein said carrying device is selected from thegroup consisting of a hollow needle, a guide wire, a balloon catheter,an ultrasound catheter, an introducer sheath, and a balloon angioplastycatheter.
 24. The apparatus of claim 11 wherein said body-insertabledevice comprises a catheter.